Transformed cell, method of screening anti-aging agent and anti-aging agent
a technology of transformed cells and anti-aging agents, applied in the field of new transformed cells, can solve problems such as long-term unclearness
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reference example 1
(1) Culturing of Cell
[0128]In this reference example, using a human normal fibroblast WI-38 (purchased from JCRB cell bank), confirmation was made on the differences in various aging indexes between active proliferating young cell which carried out 42 times of cell division after collection of the cell and its senescent cell after 61 times of cell division.
[0129]In this case, the aforementioned cells were cultured using 10% fetal calf serum (FCS)-containing DMEM (Dulbecco's modified Eagle's medium), and their sub-culturing was carried out at a ratio of 1:4. In that case, one sub-culturing was defined as 2 PD. According to this, a cell of from 30 to 40 PD was used as the young cell, and a cell of 60 PD as the senescent cell.
(2) Detection of Cellular Senescence-specific β-galactosidase (Senescence-associated β-galactosidase) Activity
[0130]The cellular senescence-specific β-galactosidase activity was detected by the method of Dimri et al. (Proc. Natl. Acad. Sci. USA, 92: 9363-9363, 199...
example 1
(1) Effect of p38 Protein Inhibitor in Human Normal Fibroblast
[0141]In the example, effect of a p38 protein inhibitor SB203580 was compared using the same young cell (42 times of division) and senescent cell (61 times of division) of the human normal fibroblast WI-38 used in Reference Example 1. Specifically, the same procedure of the following Example 2 (2) was repeated, except that a young cell (42 times of division) and a senescent cell (61 times of division) of the human normal fibroblast WI-38 cultured in the same manner in Reference Example 1(1) were used instead of the transformed cell.
[0142]The results are shown in FIG. 3. In FIG. 3, the polygonal line a is cell growth curve of the young cell in the presence of SB203580, the polygonal line b is cell growth curve of the young cell in the absence of SB203580, the polygonal line c is cell growth curve of the senescent cell in the presence of SB203580 and the polygonal line d is cell growth curve of the senescent cell in the abs...
example 2
(1) Preparation of Transformed Cell of the Present Invention
[0146]In the example, the transformed cell of the present invention was prepared in accordance with the procedure described as follows, by introducing a human MKK6 gene coding for the human MKK6 protein, or an MKK6EE gene coding for a constitutive active type mutant of the MKK6 (to be referred to as MKK6EE protein hereinafter), into a young cell of the human normal fibroblast WI-38 by a gene introduction method which uses a retrovirus.
[0147]In this connection, the MKK6 protein is an MAPK kinase (MAPKK) which phosphorylates and activates p38 protein. Also, the MKK6EE protein is a mutant of the MKK6 protein in which serine at the 207th position (Ser 207) and threonine at the 211th (Thr 211) are respectively substituted with glutamic acid (Glu; E) (Mol. Cell. Biol., 16: 1247-1255, 1996).
[0148]The MKK6 gene was obtained by amplifying a DNA fragment of about 1 kbp by a PCR method which uses a plasmid pSRα-MKK6 (J. Biol. Chem., 2...
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